Ginsenoside composition with function of regulating blood viscosity as well as preparation method and application of ginsenoside composition

Abstract

The invention relates to a ginsenoside composition with an effect of regulating blood viscosity as well as a preparation method and application of the ginsenoside composition, and belongs to the technical field of biology. The invention provides a ginsenoside composition. The ginsenoside composition comprises at least two of ginsenoside Rc, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg6, ginsenoside F4, ginsenoside S-Rg3, ginsenoside R-Rg3, ginsenoside PPT, ginsenoside Rk1, ginsenoside Rg5 and ginsenoside Rh2. The ginsenoside composition provided by the invention has the efficacy of improving body weight rise, liver injury, lipid metabolism disorder, insulin level rise and oxidative stress caused by high-fat and high-sugar diet, and has the efficacy of improving blood viscosity rise, blood coagulation time reduction and blood coagulation factor level improvement caused by acute blood stasis; the effects of improving liver injury, blood fat, blood sugar and lipid metabolism disorder, regulating blood viscosity and resisting oxidation can be achieved, and the compound can be expected to be developed into an active raw material to be applied to the fields of biological medicine, cosmetics, food, health care products and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a ginsenoside composition having the effect of regulating blood viscosity, its preparation method and application. Background Art

[0002] Blood viscosity diseases include abnormal blood glucose, abnormal blood lipids, non-alcoholic fatty liver, overweight, hypertension, atherosclerotic cardiovascular and cerebrovascular diseases, etc. Their incidence rates remain high, which is a worldwide problem. The long-term abnormal levels of sugar, lipids, and blood pressure will damage the whole body organs and lead to the gradual decline of their functions.

[0003] The therapeutic drugs for hyperlipidemia mainly include statins, resin drugs, niacin, ezetimibe, etc. However, most of these drugs have side effects. Therefore, increasing the research and development and application of plant functional foods has become a promising and potentially beneficial method for preventing and treating chronic metabolic diseases including hyperlipidemia. However, there is currently no plant-derived functional composition that uses food raw materials, has a clear mass ratio of active ingredients in the formula, has a significant effect on regulating blood viscosity, and at the same time has the effects of improving blood lipids, blood glucose, liver injury, and antioxidant. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a ginsenoside composition having the effect of regulating blood viscosity, its preparation method and application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a ginsenoside composition, which comprises at least two of ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2.

[0007] The present invention obtains a ginsenoside composition containing various ginsenosides by heating the extract of Panax notoginseng stems and leaves and hawthorn juice.

[0008] As a preferred embodiment of the ginsenoside composition of the present invention, the ginsenoside composition comprises at least four of ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2.

[0009] As a preferred embodiment of the ginsenoside composition of the present invention, the ginsenoside composition comprises at least eight of ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2.

[0010] As a preferred embodiment of the ginsenoside composition of the present invention, the ginsenoside composition comprises ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2.

[0011] As a preferred embodiment of the ginsenoside composition of the present invention, the mass ratio of ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2 in the ginsenoside composition is Rc:Rb2:Rb3:Rd:Rg6:F4:S-Rg3:R-Rg3:PPT:Rk1:Rg5:Rh2 = (0.01 - 1.0):(0.0 - 0.5):(0.03 - 1.0):(0.0 - 0.1):(0.1 - 0.7):(0.5 - 1.8):(0.5 - 1.9):(1.0 - 2.8):(0.1 - 0.5):(0.15 - 1.0):(0.1 - 0.8):(0.1 - 0.4).

[0012] As a preferred embodiment of the ginsenoside composition of the present invention, the mass ratio of ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2 in the ginsenoside composition is Rc:Rb2:Rb3:Rd:Rg6:F4:S-Rg3:R-Rg3:PPT:Rk1:Rg5:Rh2 = (0.01 - 0.30):(0 - 0.02):(0.03 - 0.31):(0.00 - 0.01):(0.39 - 0.46):(0.95 - 1.16):(0.97 - 1.20):(1.32 - 2.24):(0.29 - 0.35):(0.41 - 0.68):(0.29 - 0.43):(0.26 - 0.39).

[0013] In a second aspect, the present invention provides a method for preparing the above ginsenoside composition, which comprises mixing the extract of Panax notoginseng stems and leaves with hawthorn juice, followed by heating, concentration and drying to obtain the ginsenoside composition.

[0014] The present invention develops a method for efficiently converting the extract of Panax notoginseng stems and leaves into ginsenosides by using hawthorn juice. The extract of Panax notoginseng stems and leaves can be converted into ginsenosides through heat treatment, which has the advantages of high efficiency, convenience and rapidity. The ginsenoside composition prepared by this method contains a variety of ginsenosides, including ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2.

[0015] As a preferred embodiment of the preparation method of the present invention, the ratio of the Panax notoginseng stem and leaf extract to the hawthorn juice is Panax notoginseng stem and leaf extract: hawthorn juice = (1 - 5) kg: (6 - 14) L.

[0016] As a preferred embodiment of the preparation method of the present invention, the conditions for the heat treatment are heating at 70 - 100 °C for 60 - 240 min.

[0017] Under specific ratio ranges and heating conditions, the ginsenoside composition prepared by the present invention has excellent effects in improving liver injury, blood lipids, blood glucose, and lipid metabolism disorders, regulating blood viscosity, and antioxidation.

[0018] As a preferred embodiment of the preparation method of the present invention, the ratio of the Panax notoginseng stem and leaf extract to the hawthorn juice is Panax notoginseng stem and leaf extract: hawthorn juice = 1 kg: (6 - 8) L.

[0019] As a preferred embodiment of the preparation method of the present invention, the conditions for the heat treatment are heating at 90 °C for 180 - 210 min.

[0020] Under specific ratio ranges and heating conditions, the ginsenoside extract prepared by the present invention has the best effects in improving liver injury, blood lipids, blood glucose, and lipid metabolism disorders, regulating blood viscosity, and antioxidation.

[0021] As a preferred embodiment of the preparation method of the present invention, the hawthorn juice is concentrated hawthorn juice, and the concentrated hawthorn juice is diluted 2 - 6 times before mixing with the Panax notoginseng stem and leaf extract.

[0022] As a preferred embodiment of the preparation method of the present invention, the main active substance in the Panax notoginseng stem and leaf extract is saponin, and the mass of the saponin accounts for at least 5% of the total mass of the Panax notoginseng stem and leaf extract. The present invention does not specifically limit the saponin content in the used Panax notoginseng stem and leaf extract, and only needs to determine that the saponin content in the Panax notoginseng stem and leaf extract ≥ 5%. Moreover, the higher the saponin content in the Panax notoginseng stem and leaf extract, the better the efficacy of the prepared ginsenoside composition.

[0023] As a preferred embodiment of the preparation method of the present invention, the mass of the saponin is 6.78 - 7.32% of the total mass of the Panax notoginseng stem and leaf extract.

[0024] In the third aspect, the present invention provides the application of the above ginsenoside composition in the preparation of at least one of the following products:

[0025] (1) Products for improving liver injury;

[0026] (2) Products for improving blood lipids;

[0027] (3) Products for improving blood glucose;

[0028] (4) Products for improving lipid metabolism disorders;

[0029] (5) Products for regulating blood viscosity;

[0030] (6) Antioxidant products.

[0031] The ginsenoside composition provided by the present invention has the effects of improving weight gain, liver injury, lipid metabolism disorders, rising insulin levels, and oxidative stress caused by high-fat and high-sugar diets, and has the effects of improving the rising blood viscosity, reducing coagulation time, and increasing the levels of coagulation factors caused by acute blood stasis. It can achieve the effects of improving liver injury, blood lipids, blood sugar, and lipid metabolism disorders, regulating blood viscosity, and antioxidant, and is expected to be developed into an active ingredient for application in the fields of biomedicine, cosmetics, food, and health products.

[0032] As a preferred embodiment of the application described in the present invention, the product for improving liver injury is a product for improving liver injury caused by high sugar and high fat.

[0033] As a preferred embodiment of the application described in the present invention, the ginsenoside composition has the effects of alleviating liver tissue injury and reducing lipid accumulation.

[0034] As a preferred embodiment of the application described in the present invention, the ginsenoside composition has the effect of inhibiting insulin secretion.

[0035] As a preferred embodiment of the application described in the present invention, the ginsenoside composition has the effect of regulating lipid metabolism disorders.

[0036] As a preferred embodiment of the application described in the present invention, the ginsenoside composition has the effect of improving oxidative stress induced by high-fat and high-sugar diets in rats, thereby playing an antioxidant role.

[0037] As a preferred embodiment of the application described in the present invention, the ginsenoside composition has the effects of regulating the low, medium, and high shear viscosities of whole blood and plasma, improving blood coagulation function, and improving vascular endothelial damage.

[0038] As a preferred embodiment of the application described in the present invention, the products include drugs, foods, health products, cosmetics, and biological products.

[0039] Fourthly, the present invention provides the application of the above-mentioned ginsenoside composition in the preparation of products for improving liver injury, blood lipids, blood sugar, and lipid metabolism disorders, regulating blood viscosity, and antioxidant.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] 1. The present invention obtains a composition containing ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2 by heating the extract of Panax notoginseng stems and leaves and hawthorn juice.

[0042] 2. The present invention develops a method for efficiently converting the extract of Panax notoginseng stems and leaves into ginsenosides using hawthorn juice. By heating treatment, the extract of Panax notoginseng stems and leaves can be converted into ginsenosides, which has the advantages of high efficiency, convenience and speed. The ginsenoside composition prepared by this method contains a variety of ginsenosides, including ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2.

[0043] 3. The ginsenoside composition provided by the present invention has the effects of improving weight gain, liver injury, lipid metabolism disorder, insulin level increase and oxidative stress caused by high-fat and high-sugar diet, and has the effects of improving the increase in blood viscosity, reduction of coagulation time and increase in coagulation factor level caused by acute blood stasis. It can regulate blood viscosity, improve liver injury, blood lipid, blood sugar and lipid metabolism disorder, and has antioxidant effects, and is expected to be developed into active raw materials for application in the fields of biomedicine, cosmetics, food and health products, etc. Description of the Drawings

[0044] Figure 1 It is the detection result of the saponin content in the extract of Panax notoginseng stems and leaves from different sources in Example 1 of the present invention;

[0045] Figure 2 It is the liquid chromatogram of ginsenoside Rb1, Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2 standards in Effect Example 1 of the present invention;

[0046] Figure 3 It is the liquid chromatogram of the ginsenoside composition of Example 16 in Effect Example 1 of the present invention;

[0047] Figure 4 It is the liquid chromatogram of the ginsenoside composition of Example 17 in Effect Example 1 of the present invention;

[0048] Figure 5 It is the liquid chromatogram of the ginsenoside composition of Example 18 in Effect Example 1 of the present invention;

[0049] Figure 6 It is the result of the influence of different dilution multiples of hawthorn juice on the conversion rate and conversion multiple of the extract of Panax notoginseng stems and leaves into ginsenosides in Effect Example 1 of the present invention;

[0050] Figure 7Results of the effects of different ratios of material liquid on the conversion rate and conversion multiple of Panax notoginseng stem and leaf extract into ginsenosides in Example 1 of the present invention;

[0051] Figure 8 Results of the effects of different heating times on the conversion rate and conversion multiple of Panax notoginseng stem and leaf extract into ginsenosides in Example 1 of the present invention;

[0052] Figure 9 Statistical results of the body weight and body mass index of rats in different treatment groups in Example 2 of the present invention;

[0053] Figure 10 HE staining results of liver tissues of rats in different treatment groups in Example 2 of the present invention, scale bar is 50μm;

[0054] Figure 11 Oil red O staining results of liver tissues of rats in different treatment groups in Example 2 of the present invention, scale bar is 50μm;

[0055] Figure 12 Detection results of serum liver enzyme levels of rats in different treatment groups in Example 2 of the present invention;

[0056] Figure 13 Detection results of serum lipid levels of rats in different treatment groups in Example 2 of the present invention;

[0057] Figure 14 Detection results of fasting insulin levels of rats in different treatment groups in Example 2 of the present invention;

[0058] Figure 15 Detection results of serum SOD and MDA levels of rats in different treatment groups in Example 2 of the present invention;

[0059] Figure 16 Detection results of whole blood viscosity at low and medium shear rates of rats in different treatment groups in Example 3 of the present invention;

[0060] Figure 17 Detection results of whole blood viscosity and plasma viscosity at medium and high shear rates of rats in different treatment groups in Example 3 of the present invention;

[0061] Figure 18 Detection results of blood coagulation function of rats in different treatment groups in Example 3 of the present invention;

[0062] Figure 19 Detection results of blood coagulation factor levels of rats in different treatment groups in Example 3 of the present invention;

[0063] In the above figures, the same lowercase letters between two groups indicate significant differences between them, p < 0.05. Detailed implementation manners

[0064] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0065] For other materials, reagents, etc. used in the examples, comparative examples, and effect examples, unless otherwise specified, they can all be obtained commercially.

[0066] Four kinds of Panax notoginseng stem and leaf extracts were respectively purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd. (20220901), Guangdong Qingyunshan Pharmaceutical Co., Ltd. (20221001), Perfect (China) Co., Ltd. (20221107), and Perfect (China) Co., Ltd. (20221101).

[0067] HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences, with the catalog number SCSP-510.

[0068] The hawthorn juice of the present invention is not limited to its source. The hawthorn juice used in the following examples and effect examples was purchased from Hebei Peiran Century Bio-Food Co., Ltd., with the catalog number 20220903.

[0069] Four-week-old Sprague-Dawley rats, half male and half female, weighing 180 ± 20 g (certificate number: 210726230102814165), were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China), (license number: SXXK (Liao) 2020-001).

[0070] The test kits for aspartate aminotransferase (AST) (C010-2-1), alanine aminotransferase (ALT) (C009-2-1), triglyceride (TG) (A110-1-1), total cholesterol (TC) (A111-1-1), low-density lipoprotein cholesterol (LDL-C) (A113-1-1), and high-density lipoprotein cholesterol (HDL-C) (A112-1-1) all came from Nanjing Jiancheng Bioengineering Institute; the enzyme-linked immunosorbent assay kits for rat insulin (INS) (AD3223Ra), rat alpha granule membrane protein (GMP-140) (AD2823Ra), rat 6-keto-prostaglandin F1α (6-keto-PGF1α) (Rat0243), rat thromboxane B2 (TXB2) (Rat0521), and rat von Willebrand factor (vWF) (AD2908Ra) all came from Beijing Andy Huatai Technology Co., Ltd.

[0071] Specific operation steps of the MTT experiment: Collect cells in the logarithmic phase and adjust the cell suspension concentration to 1×10 5Cells were seeded at a density of 1×10⁴ cells / mL, and 100 μL of cell suspension was added to each well of a 96-well plate until the cell monolayer covered the bottom of the well and adhered well. Then, extracts of Panax notoginseng stems and leaves with different concentration gradients were added, 100 μL per well, with 3 replicates per group. A control group and different dose groups of Panax notoginseng stems and leaves extracts were set up. The cells were incubated at 37 °C in a 5% CO₂ incubator for 24 h. Four hours before the end of the incubation period, 10 μL of MTT solution (0.5% MTT) was added to each well and the cells were further cultured. The culture was terminated, and the culture medium in the wells was carefully aspirated. Then, 100 μL of DMSO was added to each well, and the plate was incubated at 37 °C until the crystals were completely dissolved. The OD value was measured at 490 nm using an ELISA reader.

[0072] Specific operation steps of Oil Red O staining experiment: HepG2 cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37 °C in a 5% CO₂ environment. When the cell density reached 70% - 80%, the cells were digested with trypsin and dispersed to form a cell suspension with a density of 5×10 4 cells / mL. Then, 100 μL of the cell suspension was seeded into each well of a 12-well plate. When the cells adhered and grew to 70% - 80%, the cells were treated with DMEM medium containing 0.125 mmol / L oleic acid and cultured for another 24 h. Then, the supernatant was discarded, and the cells were washed twice with 0.5 mL of PBS each time. Next, 0.5 mL of 4% paraformaldehyde was added to each well and allowed to stand at room temperature for 15 min, after which it was discarded, and the cells were washed twice with 0.5 mL of PBS each time. Then, 0.5 mL of Oil Red O working solution was added to each well and stained for 15 min, after which it was discarded. After washing twice with 0.5 mL of PBS, the cell staining was photographed under a microscope.

[0073] HPLC detection conditions (for ginsenoside quantification): HPLC, the equipment used was the L-2200 high-performance liquid chromatograph of Hitachi, Ltd., and the chromatographic column was the Agela Promosil C18 chromatographic column of Tianjin Bonna Aijier Technology Co., Ltd. The column temperature was 35 °C, the detection wavelength was 203 nm, mobile phase A was acetonitrile, and mobile phase B was water. The flow rate was 1.0 mL / min, and the injection volume was 20 μL. The elution conditions were as follows: from 0 to 10 min, 22 - 27% of A; from 10 to 15 min, 27 - 30% of A; from 15 to 35 min, 30 - 32% of A; from 35 to 40 min, 32 - 35% of A; from 40 to 43 min, 35 - 40% of A; from 43 to 48 min, 40 - 46.9% of A; from 48 to 50 min, 46.9 - 47.6% of A; from 50 to 65 min, 47.6 - 48.5% of A; from 65 to 68 min, 48.5 - 53% of A; from 68 to 73 min, 53 - 65% of A; from 73 to 78 min, 65 - 85% of A; from 78 to 83 min, 85% of A; from 83 to 85 min, 85 - 22% of A; from 85 to 95 min, 22% of A; from 95 to 100 min, 22% of A.

[0074] HPLC-MS technology (for ginsenoside qualitative analysis). The equipment is Dionex UltiMate 3000 mass spectrometer from Thermo Fisher Scientific (China) Co., Ltd. The detection conditions are as follows:

[0075] (1) HPLC detection conditions: Use Cica MERCK RP18 (2.0 mm × 100 mm, 3 μm) chromatographic column, column temperature 30 °C, mobile phase is acetonitrile (A) - 0.1% formic acid water (B), gradient elution is adopted, and the elution conditions are 0 - 3 min, 19% A; 3 - 6 min, 22% A; 6 - 9 min, 30% A; 9 - 16 min, 35% A; 16 - 18 min, 55% A; 18 - 22 min, 55% A; 22 - 26 min, 70% A; 26 - 19 min, 90% A; 29 - 29.1 min, 19% A; 29.1 - 32 min, 18% A. The volume flow rate is 0.25 mL / min; the column temperature is 35 °C; the sample temperature is 19 °C; the detection wavelength is 203 nm; the injection volume is 5 μL;

[0076] (2) MS detection conditions: The ion source is electrospray ionization source (ESI), the capillary temperature is 300 °C, the voltage is 3500 V, the collision gas is argon (purity ≥ 99.999%), the auxiliary gas is nitrogen (purity ≥ 99.999%), the auxiliary gas flow rate is 10 L / min, the auxiliary gas temperature is 350 °C, and the sprayer gas pressure is 275.8 kPa. The full scan range is m / z 50 - 2000; the MS2 scan range is m / z 50 - 2000.

[0077] Data statistical method: Use Graphpad Prism 8.0.2 software for significant statistical analysis. Through the one-way ANOVA (and nonparametic or mixed) analysis method under the Column analysis column, and in the post hoc test, Sidak for multiple comparisons is used for variance analysis. After making a bar chart, the first group on the left is marked with the lowercase letter a, and from left to right, they are marked as a, b, c, d... j in turn. The presence of the same lowercase letter between two groups indicates a significant difference between them (p < 0.05), otherwise it indicates no significant difference between them (p > 0.05).

[0078] The technologies not described in detail in the following examples and effect examples are all common technologies in this field, and reference can be made to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Press), "Molecular Biology Experiments (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiments" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0079] Example 1

[0080] In this embodiment, in order to detect the saponin content in the Panax notoginseng stem and leaf extract, the saponin content of the Panax notoginseng stem and leaf extracts from different sources was determined by colorimetry. The specific scheme is as follows:

[0081] Four kinds of Panax notoginseng stem and leaf extracts from different sources and batches were purchased through commercial channels, and the saponin content was determined by colorimetry respectively. The results are shown in Figure 1 and Table 1.

[0082] Table 1 Determination results of saponin content in different Panax notoginseng stem and leaf extracts

[0083]

[0084] As Figure 1 and shown in Table 1, the saponin content of the four kinds of Panax notoginseng stem and leaf extracts is 6.78 - 7.32%.

[0085] Examples 2 - 5

[0086] Examples 2 - 5 respectively provide a ginsenoside composition with the function of regulating blood viscosity and a preparation method thereof. The preparation method is to mix the ginsenoside composition with hawthorn juice and then perform heating, concentration and drying treatments to obtain the ginsenoside composition. The parameter conditions of the above steps are set in Table 2, and the selected Panax notoginseng stem and leaf extract is the second group of Panax notoginseng stem and leaf extracts in Example 1.

[0087] Table 2 Parameter condition settings for the preparation methods of different ginsenoside compositions

[0088]

[0089] Examples 6 - 9

[0090] Examples 6 - 9 respectively provide a ginsenoside composition with the function of regulating blood viscosity and a preparation method thereof. The preparation method is to mix the ginsenoside composition with hawthorn juice and then perform heating, concentration and drying treatments to obtain the ginsenoside composition. The parameter conditions of the above steps are set in Table 3, and the selected Panax notoginseng stem and leaf extract is the second group of Panax notoginseng stem and leaf extracts in Example 1.

[0091] Table 3 Parameter condition settings for the preparation methods of different ginsenoside compositions

[0092]

[0093] Examples 10 - 15

[0094] Examples 10 - 15 respectively provide a ginsenoside composition with the function of regulating blood viscosity and its preparation method. The preparation method is to mix the ginsenoside composition with hawthorn juice, followed by heating, concentration, and drying to obtain the ginsenoside composition. The parameter conditions for the above steps are set as shown in Table 4, and the selected Panax notoginseng stem and leaf extract is the second group of Panax notoginseng stem and leaf extract in Example 1.

[0095] Table 4 Parameter Conditions for the Preparation Methods of Different Ginsenoside Compositions

[0096]

[0097] Example 16

[0098] Example 16 provides a ginsenoside composition with the function of regulating blood viscosity and its preparation method. According to the ratio of Panax notoginseng stem and leaf extract: hawthorn juice = 1 kg: 12 L, 0.1 kg of Panax notoginseng stem and leaf extract is mixed with 1.2 L of hawthorn juice, followed by heating, concentration, and drying to obtain the ginsenoside composition. The remaining parameter settings are carried out according to those in Example 14.

[0099] Example 17

[0100] Example 17 provides a ginsenoside composition with the function of regulating blood viscosity and its preparation method. According to the ratio of Panax notoginseng stem and leaf extract: hawthorn juice = 1 kg: 12 L, 1 kg of Panax notoginseng stem and leaf extract is mixed with 12 L of hawthorn juice, followed by heating, concentration, and drying to obtain the ginsenoside composition. The remaining parameter settings are carried out according to those in Example 14.

[0101] Example 18

[0102] Example 18 provides a ginsenoside composition with the function of regulating blood viscosity and its preparation method. According to the ratio of Panax notoginseng stem and leaf extract: hawthorn juice = 1 kg: 12 L, 5 kg of Panax notoginseng stem and leaf extract is mixed with 60 L of hawthorn juice, followed by heating, concentration, and drying to obtain the ginsenoside composition. The remaining parameter settings are carried out according to those in Example 14.

[0103] Effect Example 1

[0104] Characterize the ginsenoside compositions obtained in Examples 16 - 18. The specific scheme is as follows:

[0105] 1. Mix the Panax notoginseng stem and leaf extract, the ginsenoside composition obtained in Examples 16 - 18 with methanol respectively to prepare solutions of 100 μg / mL, and conduct quantitative and qualitative analysis of the ginsenosides contained in the ginsenoside composition by HPLC and HPLC-MS techniques. At the same time, mix the reference standards of ginsenoside Rb1, Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2 in a mass ratio of 1:1 and conduct HPLC detection to obtain a standard curve. The results are shown in Figures 2 - 5 and Table 5.

[0106] Table 5 Qualitative and Quantitative Results of Ginsenosides in Different Ginsenoside Compositions

[0107]

[0108] As Figures 2 - 5 and shown in Table 5, reducing or increasing the dosage of the Panax notoginseng stem and leaf extract under the optimal parameter conditions can obtain a ginsenoside composition containing rare ginsenosides Rc, Rb2, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2, indicating that the preparation method of the present invention can successfully prepare the ginsenoside composition.

[0109] 2. Conduct qualitative and quantitative analysis of the ginsenoside compositions obtained in Examples 2 - 15 according to the above HPLC-MS method, and calculate the conversion rate and conversion multiple of ginsenosides. The content of the saponins after conversion is all converted to calculate based on the Panax notoginseng stem and leaf extract (g). The results are shown in Figures 6 - 8 , and the calculation formula is as follows:

[0110]

[0111] In the formula, the raw material refers to the Panax notoginseng stem and leaf extract before conversion, the converted refers to the ginsenoside composition, and the rare saponins refer to ginsenosides Rc, S-Rg3, R-Rg3, Rk1 and Rg5;

[0112]

[0113] In the formula, the raw material refers to the Panax notoginseng stem and leaf extract before conversion, the converted refers to the ginsenoside composition, and the prototype saponins refer to ginsenosides Rb1, Rb2 and Rb3.

[0114] As Figure 6 shown, diluting the concentrated hawthorn juice by 0 - 4 times can effectively improve the conversion of the Panax notoginseng stem and leaf extract into ginsenosides, thereby increasing the content of rare ginsenosides in the ginsenoside composition. Therefore, choosing to mix the concentrated hawthorn juice diluted by 0 - 4 times with the Panax notoginseng stem and leaf extract can effectively improve the conversion efficiency of the Panax notoginseng stem and leaf extract. Among them, the conversion efficiency of the concentrated hawthorn juice diluted 2 times is the best.

[0115] As Figure 7 shown, as the proportion of hawthorn juice increases, the conversion rate of Panax notoginseng stem and leaf extract also increases, while the conversion multiple shows a trend of first increasing and then decreasing. Therefore, selecting a material-liquid ratio of 1:2 to 1:10 can effectively improve the conversion efficiency of Panax notoginseng stem and leaf extract. The best material-liquid ratio is 1:8, followed by 1:6 and 1:10.

[0116] As Figure 8 shown, before and at 180 min, as the heating time increases, the conversion rate and conversion multiple of Panax notoginseng stem and leaf extract also increase. After 180 min, the conversion rate and conversion multiple of Panax notoginseng stem and leaf extract decrease. Therefore, selecting a heating time of 60 - 240 min can effectively improve the conversion efficiency of Panax notoginseng stem and leaf extract. The conversion efficiency is the best when the heating time is 180 min.

[0117] Effect Example 2

[0118] To verify the efficacy of the ginsenoside composition of Example 18, a glycolipid metabolism disorder model was constructed to evaluate the efficacy of the ginsenoside composition. The specific scheme is as follows:

[0119] 1. Experimental content.

[0120] SD rats were fed with ordinary feed for 1 week for ordinary feed adaptation. Rats with similar body weights were selected for the experiment. Except for the normal control group, the remaining groups were fed with high-fat and high-sugar feed for 4 weeks. Then, intragastric administration was started according to the grouping in Table 6 for 4 consecutive weeks. During the administration period, high-fat and high-sugar feed was normally fed. After the administration ended, samples were collected to detect the corresponding indexes. There were 6 rats in each treatment group, with 3 female and 3 male rats in each treatment group.

[0121] Table 6 Experimental grouping and its administration treatment

[0122]

[0123] 2. Index determination.

[0124] (1) Measure the body weights of rats in each group after the experiment ended, and calculate their body mass index (body mass index BMI = body weight ÷ square of body length, the unit of body weight is kg, and the unit of body length is m). The results are shown in Figure 9 .

[0125] As Figure 9 shown, compared with group C, the body weight and BMI index of group M were significantly increased, and the body weights of groups SSH, SSZ-40, SSZ-80 and TAX+MET were significantly decreased compared with that of group M, indicating that the ginsenoside composition of the present invention has a certain efficacy in weight loss.

[0126] (2) After the experiment, the rats were sacrificed and their liver tissues were collected for HE staining and Oil Red O staining. The results are shown in Figures 10 - 11 .

[0127] As Figure 10 shown, the hepatic tissue structure of group C was basically normal. The hepatocyte structure in the visual field was complete, with clear outlines, round cell nuclei, and abundant cytoplasm. There was no necrosis or degeneration. The hepatic sinusoids were of uniform size without dilation, and the hepatic cords were arranged neatly and closely. The hepatic tissue structure of group M was severely abnormal with unclear outlines. Some hepatocytes were necrotic, and there was severe fatty degeneration of a large area of hepatocytes in the visual field. Round lipid droplets of different sizes could be seen in the cytoplasm. After drug intervention in groups SQ, SZ, SSH, SSZ-20, SSZ-40, SSZ-80, TAX, and TAX-MET, there were improvements. In the visual field, partial fatty degeneration of hepatocytes was observed, and round lipid droplets of uniform size could be seen in the cytoplasm. The outlines of hepatocytes were clear, and there was no necrosis. Among them, the effects of groups SSZ-20, SSZ-40, and SSZ-80 were better than those of groups SQ, SZ, and SSH. With the increase in the dosage of SSZ administered, its effect was more obvious. The effect of SSZ-80 was close to that of the positive drugs TAX and TAX+MET groups, indicating that SSZ-80 significantly reversed liver injury.

[0128] As Figure 11 shown, compared with group C, lipid deposition was aggravated in group M, while after drug treatment in groups SQ, SZ, SSH, SSZ-20, SSZ-40, SSZ-80, TAX, and TAX-MET, the lipid droplet area was significantly reduced. Among them, the effects of groups SSZ-20, SSZ-40, and SSZ-80 were better than those of groups SQ, SZ, and SSH. With the increase in the dosage of SSZ administered, its effect was more obvious. The effect of SSZ-80 was close to that of the positive drugs TAX and TAX+MET groups, indicating that SSZ significantly improved hepatic lipid deposition.

[0129] In summary, the ginsenoside composition of the present invention can alleviate the pathological damage of liver tissue caused by high-fat and high-sugar diet and reduce lipid accumulation.

[0130] (3) After the experiment, whole blood was collected from the rats in each group, and serum was obtained by centrifugation. The operations were carried out according to the instructions of the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) kits, and the levels of AST and ALT were measured. The results are shown in Figure 12 .

[0131] The increase in the levels of hepatic enzymes AST and ALT is one of the signs of liver injury. Therefore, measuring the levels of AST and ALT can determine whether there is liver function impairment in the organism. As Figure 12As shown, compared with group C, the serum ALT and AST levels of rats fed with a high-fat and high-sugar diet were significantly increased. As expected, the liver tissue of rats fed with a high-fat and high-sugar diet was damaged.

[0132] To study the protective effect of ginsenoside composition on liver injury induced by high-fat and high-sugar diet in SD rats, after successful modeling, rats were treated with ginsenoside composition while being fed with a high-fat and high-sugar diet. Compared with the rats fed only with a high-fat and high-sugar diet (group M), the AST and ALT levels of SD rats treated with ginsenoside composition (SSZ-20, SSZ-40, and SSZ-80) were significantly decreased and showed a dose-dependent manner, suggesting that the ginsenoside composition can effectively protect the liver injury caused by a high-fat and high-sugar diet to rat liver tissue. Although the SQ, SZ, and SSH groups also reduced the secretion of ALT and AST to a certain extent, the inhibitory effect was not as good as that of the SSZ group. In addition, among the SSZ groups, the ALT and AST levels in the SSZ-80 group were the lowest, significantly lower than those in the positive drug TAX group and comparable to those in the positive drug combination group TAX+MET, indicating that the ginsenoside composition of the present invention has the efficacy of improving liver injury, and its effect is comparable to that of the positive drug combination.

[0133] (4) After the experiment, whole blood of rats in each group was collected, and serum was obtained by centrifugation. The TG, TC, HDL-C, and LDL-C levels of rats in each group were detected according to the kit instructions of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). The results are shown in Figure 13 .

[0134] As Figure 13 shown, compared with group C, the TG, TC, and LDL-C levels in group M were significantly increased, and the HDL-C level was significantly decreased (p<0.05), suggesting that lipid metabolism disorders occurred in rats fed with a high-fat and high-sugar diet, indicating that the rat model of lipid metabolism disorders induced by high-fat and high-sugar diet was successfully established. Compared with the rats fed only with a high-fat and high-sugar diet (group M), the TG, TC, and LDL-C levels of rats treated with ginsenoside composition were significantly decreased, and the HDL-C level was significantly increased. As the SSZ administration dose increased, the changing trends of the secretion levels of TG and HDL-C showed an obvious dose-dependent manner. Compared with the SQ, SZ, and SSH dose groups, the SSZ-80 group showed better regulatory effects in the regulation of TG, TC, and HDL-C, and was significantly better than the positive drug TAX group and the TAX+MET group (TG, LDL-C). The above results indicate that the ginsenoside composition of the present invention has the efficacy of regulating lipid metabolism disorders.

[0135] (5) After the experiment, whole blood of rats in the fasting state was collected, and serum was obtained by centrifugation. The fasting insulin levels of each group were detected according to the instructions of the insulin kit. The results are shown in Figure 14 .

[0136] As Figure 14 shown, the serum insulin level in group C was significantly lower than that in group M, indicating that feeding with a high-fat and high-sugar diet could lead to an increase in the fasting insulin level of rats. Compared with the rats fed only with a high-fat and high-sugar diet (group M), the insulin levels of the rats treated with ginsenoside composition (SSZ-20, SSZ-40, and SSZ-80) were significantly decreased. With the increase in the dosage of the ginsenoside composition administered to the rats, the decrease in rat insulin showed a dose-dependent relationship. Among them, the inhibitory effect of the SSZ group was significantly better than that of the SQ group, SZ group, and SSH group, indicating that the ginsenoside composition generated by the thermal conversion of the stem and leaf extract of Panax notoginseng in hawthorn juice had a better blood glucose-regulating effect than the SQ group, SZ group, and SSH group. Furthermore, the inhibitory effect of the SSZ-80 group on insulin secretion level was better than that of the positive drugs TAX group and TAX+MET group. From the above, it can be seen that the ginsenoside composition of the present invention has a good effect of regulating insulin level.

[0137] (6) After the experiment, whole blood of rats in each group was collected, and serum was obtained by centrifugation. The SOD and MDA levels in the rat serum were detected according to the instructions of the superoxide dismutase (SOD) and malondialdehyde (MDA) kits. The results are shown in Figure 15 .

[0138] MDA is a lipid peroxide, and its content can reflect the level of lipid peroxidation; SOD can scavenge oxygen free radicals and is an important factor in balancing oxidation and antioxidation. As Figure 15 shown, compared with group C, the MDA level in group M was significantly increased, and the SOD level was significantly decreased (p<0.05), indicating that high-fat and high-sugar feeding could damage the endogenous antioxidant system of rats and cause oxidative stress. Compared with the rats fed only with a high-fat and high-sugar diet (group M), treatment with ginsenoside composition (SSZ-20, SSZ-40, and SSZ-80) could significantly increase the SOD level in the rat serum and decrease the MDA level, and could improve the oxidative stress induced by a high-fat and high-sugar diet, showing a dose-dependent relationship (p<0.05). Compared with the SQ, SZ groups, and SSH group, the SSZ-20, SSZ-40, and SSZ-80 groups showed better antioxidant effects, and their antioxidant ability was comparable to that of the positive drug TAX+MET group. From the above, it can be seen that the ginsenoside composition of the present invention has a good antioxidant effect and can improve the oxidative stress caused by a high-fat and high-sugar diet.

[0139] Effect Example 3

[0140] To verify the efficacy of the ginsenoside composition in Example 18, an acute blood stasis model was constructed to evaluate the efficacy of the ginsenoside composition. The specific protocol is as follows:

[0141] 1. Experimental content.

[0142] SD rats were adaptively fed with ordinary feed for 1 week. Rats with similar body weights were selected for the experiment. They were intragastrically administered according to the groups and doses set in Table 6 every day for 8 consecutive days. 2 hours after the last administration, except for the control group which was subcutaneously injected with normal saline, the remaining rats were subcutaneously injected with 0.1 v / v% adrenaline hydrochloride solution (0.8 mL / kg). 2 hours after injection, the control group was not treated, and the remaining rats were all subjected to ice-water bath swimming at 0 - 4°C for 5 minutes. 2 hours later, they were injected with 0.1 v / v% adrenaline hydrochloride solution (0.8 mL / kg) again. After the model was established, the rats in each group were fasted but allowed to drink water. The next day, samples were collected to detect the corresponding indicators. There were 6 rats in each treatment group, with 3 female and 3 male rats in each treatment group.

[0143] 2. Index determination.

[0144] (1) After the experiment, the whole blood of rats in each group was collected, added with an appropriate amount of heparin for anticoagulation, and the whole blood viscosity and plasma viscosity were measured using the MVIS - 2035 automatic blood rheology analysis system. The results are shown in Figures 16 - 17 .

[0145] As Figures 16 - 17 shown, compared with group C, the plasma viscosity and whole blood viscosity of rats in group M were significantly higher than those in the control group, indicating the successful establishment of the acute blood stasis rat model. Although groups SQ, SZ, and SSH had a certain effect on improving the plasma viscosity and whole blood viscosity of rats, the inhibitory effect was not as good as that of group SSZ - 80. The treatment groups of the ginsenoside composition (SSZ - 20, SSZ - 40, and SSZ - 80) all had the efficacy of reducing plasma and whole blood viscosity, and as the administration dose of the ginsenoside composition group gradually increased, the plasma viscosity and whole blood viscosity of rats showed a dose-dependent downward trend.

[0146] (2) After the experiment, the whole blood of rats in each group was collected, and the PT, TT, APTT, and FIB of rats in each group were detected according to the instructions of the prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) kits. The results are shown in Figure 18 .

[0147] As Figure 18As shown, compared with group C, the PT, TT, and APTT values in group M were lower than those in group C, while the FIB content was significantly higher than that in group C. The differences were statistically significant, indicating the successful establishment of the acute blood stasis model. After drug intervention, the PT, TT, and APTT values of rats in each group increased to a certain extent, and the increase in the SZZ-80 group was the largest, superior to the SQ, SZ, SSH, TAX, and TAX+MET groups. Similar regulatory effects were also shown in the regulation of FIB content, and the inhibitory effect of the SSZ-80 group on FIB content was the best.

[0148] (3) After the experiment, the whole blood of rats in each group was collected, and the operations were carried out according to the instructions of the kits for granule membrane protein 140 (GMP-140), 6-keto-prostaglandin F1α (6-keto-PGF1α), von Willebrand factor (vWF), and thromboxane B2 (TXB2). The levels of GMP-140, 6-keto-PGF1α, vWF, and TXB2 were measured, and the results are shown in Figure 19 .

[0149] As Figure 19 shown, compared with group C, the level of 6-keto-PGF1α in rats in group M was significantly decreased, and the contents of TXB2, GMP-140, and vWF were significantly increased (p<0.05), indicating that adrenaline hydrochloride combined with ice bath swimming can damage the vascular endothelium of rats and can make rats tend to form blood stasis by promoting platelet adhesion and aggregation. Compared with group M, each drug administration group can make the secretion levels of the above-mentioned coagulation factors show an obvious reverse trend, indicating that it can improve the vascular endothelium damage induced by high-fat and high-sugar. Among them, compared with the single drug dose groups (SQ, SZ groups), SSZ showed obvious advantages, superior to the positive drug metformin and the combined drug groups.

[0150] In summary, the ginsenoside composition of the present invention has the effects of improving weight gain, liver injury, lipid metabolism disorder, insulin level increase, and oxidative stress caused by high-fat and high-sugar diets, and has the effects of improving the increase in blood viscosity, decrease in coagulation time, and increase in coagulation factor levels caused by acute blood stasis, so as to achieve the effects of improving liver injury, blood lipid, blood glucose, and lipid metabolism disorder, regulating blood viscosity, and antioxidant, and can be applied in products for improving liver injury, blood lipid, blood glucose, and lipid metabolism disorder, regulating blood viscosity, and antioxidant.

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A ginsenoside composition, characterized in that, The ginsenoside composition comprises at least two of ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2.

2. The ginsenoside composition according to claim 1, characterized in that The mass ratio of ginsenoside Rc, Rb2, Rb3, Rd, Rg6, F4, S-Rg3, R-Rg3, PPT, Rk1, Rg5 and Rh2 in the ginsenoside composition is Rc:Rb2:Rb3:Rd:Rg6:F4:S-Rg3:R-Rg3:PPT:Rk1:Rg5:Rh2 = (0.01-1.0):(0.0-0.5):(0.03-1.0):(0.0-0.1):(0.1-0.7):(0.5-1.8):(0.5-1.9):(1.0-2.8):(0.1-0.5):(0.15-1.0):(0.1-0.8):(0.1-0.4).

3. The preparation method of the ginsenoside composition according to any one of claims 1-2, characterized in that, The Panax notoginseng stem and leaf extract is mixed with hawthorn juice and then subjected to heating, concentration and drying treatments to obtain the ginsenoside composition.

4. The preparation method according to claim 3, characterized in that, It includes at least one of the following (Ⅰ) to (Ⅱ): (Ⅰ) The ratio of the Panax notoginseng stem and leaf extract to hawthorn juice is Panax notoginseng stem and leaf extract:hawthorn juice = (1-5) kg:(2-14) L; (Ⅱ) The conditions for the heating treatment are heating at 70-100 °C for 60-240 min.

5. The preparation method according to claim 4, characterized in that, It includes at least one of the following (Ⅲ) to (Ⅳ): (Ⅲ) The ratio of the Panax notoginseng stem and leaf extract to hawthorn juice is Panax notoginseng stem and leaf extract:hawthorn juice = 1 kg:(6-8) L; (Ⅳ) The conditions for the heating treatment are heating at 90 °C for 180-210 min.

6. The preparation method according to claim 3, characterized in that, The hawthorn juice is concentrated hawthorn juice, and the concentrated hawthorn juice is diluted 0-6 times before mixing with the Panax notoginseng stem and leaf extract.

7. The preparation method according to claim 3, characterized in that, The main active substance in the Panax notoginseng stem and leaf extract is saponin, and the mass of the saponin accounts for at least 5% of the total mass of the Panax notoginseng stem and leaf extract.

8. Use of the ginsenoside composition according to any one of claims 1-2 in the preparation of at least one of the following products: (1) A product for improving liver injury; (2) A product for improving blood lipid; (3) A product for improving blood glucose; (4) A product for improving lipid metabolism disorder; (5) A product for regulating blood viscosity; (6) An antioxidant product.

9. The application according to claim 8, wherein The product for improving liver injury is a product for improving liver injury caused by high sugar and high fat.

10. Use of the ginsenoside composition according to any one of claims 1-2 in the preparation of a product for improving liver injury, blood lipid, blood glucose and lipid metabolism disorder, regulating blood viscosity and antioxidant.

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